Attenuation of Endoplasmic Reticulum Stress Enhances Carvacrol-Induced Apoptosis in Osteosarcoma Cell Lines

Carvacrol is a monoterpenoid phenol that has excellent antimicrobial, antiviral, and anti-inflammatory activities. It can also improve wound healing. However, few studies have explored its antitumor effect on osteosarcoma. In this report, we tried to determine the potential efficacy of carvacrol against osteosarcoma cell lines. Our data revealed that carvacrol exposure inhibited the proliferation of osteosarcoma HOS and U-2 OS cells. In addition, carvacrol exposure enhanced the levels of cleaved PARP and caspase 3 and increased annexin V-positive cells, indicating that carvacrol exposure triggers apoptosis in osteosarcoma cell lines. Furthermore, the levels of reactive oxygen species (ROS) were enhanced after carvacrol exposure and cotreatment with NAC, the ROS scavenger, decreased the levels of cleaved PARP and caspase 3, suggesting the involvement of ROS in carvacrol-induced apoptosis. Importantly, we found that carvacrol exposure triggered several protein expressions related to endoplasmic reticulum (ER) stress, including GRP78/Bip, IRE1a, PERK, and CHOP, in HOS and U-2 OS cells, indicating that carvacrol exposure could result in ER stress in these cell lines. Cotreatment with the ER stress inhibitor 4-PBA increased the levels of cleaved PARP and caspase 3 and further suppressed cellular proliferation in carvacrol-exposed osteosarcoma cell lines. Overall, the results indicate that induced ER stress can protect cells from apoptosis, but increased ROS contributes to apoptosis in carvacrol-treated cells. In this report, we first demonstrate the role of ER stress in carvacrol-induced apoptosis and suggest that ER stress could be targeted to enhance the antitumor activity of carvacrol in osteosarcoma cell lines.


Introduction
Osteosarcoma is the most common bone cancer and predominantly occurs in adolescents and children. It is characterized by the presence of malignant mesenchymal cells producing osteoid or immature bone. Most patients suffer from pain and swelling or lumps in the involved region. Osteosarcoma may form a persistent course with local and systemic disease progression and result in death if not treated. The estimated incidence rate of osteosarcoma is eight cases/million/year in children younger than 20 years of age [1,2]. The risk factors of osteosarcoma include young age, greater height, radiation treatment for other types of cancers, and Paget disease of the bone. In addition, certain inherited cancer syndromes are associated with increased risk of developing osteosarcoma, including hereditary retinoblastoma, Li-Fraumeni syndrome, Rothmund-Thomson syndrome, Bloom syndrome, Werner syndrome, and Diamond-Blackfan anemia. Some gene mutations, such used as a chemical flavoring in various foods, such as alcoholic beverages. Several studies have indicated that carvacrol has excellent activities to inhibit the growth of microbes and viruses [17,18]. In addition, it has been demonstrated that carvacrol has anti-inflammatory activities and benefits wound healing by modulating inflammatory cytokines and improving re-epithelialization, angiogenesis, and collagen deposition [19,20]. Recent literature has shown its anticancer effects in several cancer cells. Carvacrol inhibits cell proliferation and induces apoptosis in cervical cancer via mediating MEK/ERK activation [21]. Carvacrol suppresses the expressions of matrix metalloproteinase-2/9 (MMP2/9) and triggers cell cycle arrest in G2/M phase and apoptosis to inhibit colon cancer proliferation [22]. Carvacrol inhibits the proliferation of human breast cancer MCF-7 cells via p53-dependnet apoptosis [23].
Reactive oxygen species (ROS) are highly reactive molecules that have essential roles in cellular signal transduction and homoeostasis. They can modulate various biological processes, such as cell proliferation, differentiation, and angiogenesis. However, excessive levels of ROS can cause cellular damage, leading to activation of the cell death machinery [24]. ROS-dependent cell death has been shown to be involved in the antitumor effect of different chemotherapeutic agents [25]. For example, cisplatin induces apoptosis via ROS overgeneration in osteosarcoma MG-63 cell line [26], methotrexate triggers cell death by upregulating ROS production in osteosarcoma U-2 OS cells [27], and carvacrol triggers ROS-dependent apoptosis to suppress proliferation in prostate cancer [28] and lung adenocarcinoma cell lines [29].
Endoplasmic reticulum (ER) is a membrane system of cells, and it is responsible for intracellular calcium balance, protein biosynthesis, protein folding, and transporting of synthesized proteins. When misfolded and unfolding proteins accumulate in the ER lumen, ER stress occurs and unfolded protein response (UPR) is activated to modulate proper protein folding and degradation of unfolded proteins [30]. Three ER stress protein sensors, namely, IRE1, PERK, and ATF6, are involved in UPR initiation. The three ER stress sensors are normally bound by the ER resident chaperone GRP78, which maintains them in an inactive state. Under ER stress, the three ER stress sensors are released from GRP78 and thus activate downstream signaling cascades to restore ER homeostasis [31]. However, if it fails to cope with unfolded proteins, UPR triggers cell death [32]. 4-Phenylbutyric acid (4-PBA) is an FDA-approved drug for urea cycle disorders. It can also act as an inhibitor of ER stress by promoting protein folding [33].
Several studies have shown that carvacrol has excellent activities to modulate different biological processes. However, few studies have explored its antitumor effect on osteosarcoma. In this report, we tried to estimate the potential efficacy of carvacrol against osteosarcoma cell lines and reveal the roles of ROS and ER stress in this effect.

Cell Culture and Chemical
Osteosarcoma HOS and U-2 OS cells were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). HOS and U-2 OS cell lines were cultured in MEM and McCoy's 5A medium, respectively, with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 37 • C in a 5% CO 2 incubator. Carvacrol (purity ≥98%) and sodium 4-phenylbutyrate (4-PBA, purity ≥98%) were obtained from Cayman Chemical Company (Ann Arbor, MI, USA).

Cell Viability Assay
To determine cell viability, HOS and U-2 OS cells were seeded at a density of 2 × 10 4 and 2.5 × 10 4 /well, respectively, in 24-well dishes overnight. The cells were exposed to different concentrations of carvacrol for 24 or 48 h. Finally, 10 µL of MTT solution (5 mg/mL) was added to the wells, and the cells were then incubated at 37 • C for another 2 h. After that, the medium with MTT was withdrawn, and 200 µL of DMSO was used to dissolve formazan crystals. The absorbance of samples was then measured at 570 nm by a microplate reader (Molecular Devices, San Jose, CA, USA). The IC 50 values were calculated using the "Forecast" function in Microsoft Excel.

Colony Formation Assay
The cells (1.5 × 10 5 /dish) were incubated with different concentrations of carvacrol (0, 0.5, 0.6, and 0.7 mM) in the absence or presence of 4-PBA (1, 3, and 5 mM) for 16 h. After that, 500 of the exposed cells were seeded into 35 mm dishes with fresh culture medium without carvacrol. After 10 days of culture, crystal violet was used to stain colonies before counting.

Cell Cycle Analysis
Cells were exposed to carvacrol (0, 0.5, 0.6, and 0.7 mM) for 24 h. The exposed cells were collected, fixed with 100% ethanol, and stored at −20 • C overnight. After that, cells were washed twice with PBS and then incubated with a solution containing 40 µg/mL PI and 50 µg/mL RNase I in PBS for 30 min at room temperature (RT) in the dark. Finally, DNA content was examined by flow cytometry (Beckman Coulter Inc., San Jose, CA, USA).

Apoptosis Cell Assay
Apoptotic cells were determined by the annexin V/PI double staining kit (Becton Dickinson, San Jose, CA, USA). Initially, HOS and U-2 OS cells were seeded at a density of 1 × 10 5 and 1.3 × 10 5 /35 mm dish, respectively. The cells were incubated with carvacrol (0, 0.5, 0.6, and 0.7 mM) for 24 or 48 h. After that, the cells were washed, harvested, and stained with annexin V-FITC and PI according to the manufacturer's protocol and analyzed by flow cytometry (Beckman Coulter Inc., San Jose, CA, USA).

Measurement of Intracellular Reactive Oxygen Species (ROS)
The cell permeant reagent 2 ,7 -dichlorofluorescin diacetate (DCFDA) (Abcam, Cambridge, UK) was used to quantify the levels of ROS in live cell samples. Cells were exposed to 0.5 mM of carvacrol in the presence or absence of N-acetyl-L-cysteine for 24 h. These exposed cells were trypsinized, washed once by PBS, suspended, and incubated with 500 µL of PBS containing DCFDA (0.5 µM) for 20 min at 37 • C. Finally, the intracellular fluorescence was measured by flow cytometry (Beckman Coulter Inc., San Jose, CA, USA).

Statistical Analysis
All results are presented as mean ± standard error of mean (SEM) and were analyzed using Dunnett's multiple comparisons test and Sidak's multiple comparisons test for significant differences. p value < 0.05 was considered statistically significantly.

Carvacrol Treatment Suppresses Cell Viability of Osteosarcoma Cell Lines
We first examined the effects of carvacrol on viability and proliferation of human osteosarcoma cell lines using the MTT assay. As shown in Figure 1A, carvacrol exposure apparently reduced cell viabilities of osteosarcoma HOS and U-2 OS cells (IC 50 = 0.56 mM for HOS cells and 0.56 mM for U-2 OS cells at 24 h; IC 50 = 0.47 mM for HOS cells and 0.39 mM for U-2 OS cells at 48 h). In addition, colony formation assay was used to examine the effect of carvacrol on single-cell proliferative capability of osteosarcoma cell lines. The results showed that carvacrol exposure decreased the colony number at the concentrations of 0.5, 0.6, and 0.7 mM in HOS and U-2 OS cells ( Figure 1B). These results demonstrate that carvacrol could have antiproliferative effects against osteosarcoma cell lines.

Statistical Analysis
All results are presented as mean ± standard error of mean (SEM) and were analyzed using Dunnett's multiple comparisons test and Sidak's multiple comparisons test for significant differences. p value < 0.05 was considered statistically significantly.

Carvacrol Treatment Suppresses Cell Viability of Osteosarcoma Cell Lines
We first examined the effects of carvacrol on viability and proliferation of human osteosarcoma cell lines using the MTT assay. As shown in Figure 1A, carvacrol exposure apparently reduced cell viabilities of osteosarcoma HOS and U-2 OS cells (IC50 = 0.56 mM for HOS cells and 0.56 mM for U-2 OS cells at 24 h; IC50 = 0.47 mM for HOS cells and 0.39 mM for U-2 OS cells at 48 h). In addition, colony formation assay was used to examine the effect of carvacrol on single-cell proliferative capability of osteosarcoma cell lines. The results showed that carvacrol exposure decreased the colony number at the concentrations of 0.5, 0.6, and 0.7 mM in HOS and U-2 OS cells ( Figure 1B). These results demonstrate that carvacrol could have antiproliferative effects against osteosarcoma cell lines.  The cells were exposed to carvacrol (0.5, 0.6, and 0.7 mM) for 16 h. After that, 500 cells of the exposed cells were seeded into 35 mm dishes with fresh culture medium without carvacrol. After 10 days of culture, crystal violet was used to stain colonies, and the number of colonies was counted. Results are presented as mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 compared to the 0 mM group.
To examine whether apoptosis is involved in the antigrowth effect of carvacrol, populations of sub-G1 on DNA content, the marker of apoptotic cells, were estimated by flow cytometry in carvacrol-treated osteosarcoma cell lines. As shown in Figure 2A, populations of sub-G1 were significantly upregulated after carvacrol treatment (0.5-0.7 mM) for 24 h in HOS and U-2 OS cells. Next, the apoptotic effects of carvacrol were verified by annexin V/PI double staining using flow cytometry. As shown in Figure 2B, the percentage of annexin-V positive cells in carvacrol-exposed cells were significantly upregulated. Furthermore, the Life 2023, 13, 744 6 of 13 levels of cleaved caspase 3 and PARP, the apoptosis-related proteins, were examined by Western blotting. As shown in Figure 2C, carvacrol exposure for 24 h increased the levels of cleaved caspase 3 and PARP in a dose-dependent manner. Moreover, the caspase inhibitor Z-VAD-FMK decreased the levels of cleaved caspase 3 in carvacrol-treated osteosarcoma cell lines ( Figure 2D) and partly rescued the antiproliferative effect of carvacrol treatment in these cell lines ( Figure 2E). The data indicate that carvacrol exposure can trigger apoptosis in osteosarcoma cell lines. In addition, our data showed that high dose of carvacrol (0.7 mM) resulted in an increase in PI-positive but annexin V-negative cells in HOS cells ( Figure 2B), suggesting that the dose of carvacrol could induce nonapoptotic cell death in this cell line.  (C) Carvacrol exposure increases the levels of cleaved caspase 3 and cleaved PARP in osteosarcoma cell lines. The cells were exposed to carvacrol for 24 h. After that, the cells were analyzed by Western blotting using indicated antibodies by independent duplicate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 compared to the 0 mM group. (D) Caspase inhibitor decreases the levels of cleaved caspase 3 in carvacrol-treated osteosarcoma cell lines. HOS or U-2 OS cells were exposed to carvacrol (0.7 mM) for 24 h in the presence or absence of Z-VAD-FMK (10 µM). After that, the exposed cells were analyzed by Western blotting using indicated antibodies by independent duplicate experiments. (E) Caspase inhibitor partly rescues the antiproliferative effect of carvacrol treatment. HOS and U-2 OS cells were exposed to carvacrol (0.7 mM) for 48 h, and cell viability was estimated by MTT assay. Data are presented as mean ± S.E.M. of three independent experiments. ** p < 0.01, # p < 0.0001 as compared with the Z-VAD 0 µM group.

ROS Is Involved in Carvacrol-Induced Apoptosis in Osteosarcoma Cell Lines
Evidence indicates that reactive oxygen species (ROS) could regulate cell apoptosis [34]. We estimated the cellular redox status after carvacrol exposure for 24 h in osteosarcoma cell lines using DCFDA, a fluorogenic dye that assesses ROS within the cell. As shown in Figure 3A, carvacrol exposure significantly upregulated the levels of ROS in HOS and U-2 OS cells. Furthermore, coadministration of N-acetyl-L-cysteine (NAC), a ROS scavenger, resulted in attenuation of the increased cleaved caspase 3 and PARP in these cells ( Figure 3B). The results suggest that ROS may be involved in carvacrol-induced apoptosis in osteosarcoma HOS and U-2 OS cells.

ROS Is Involved in Carvacrol-Induced Apoptosis in Osteosarcoma Cell Lines
Evidence indicates that reactive oxygen species (ROS) could regulate cell apoptosis [34]. We estimated the cellular redox status after carvacrol exposure for 24 h in osteosarcoma cell lines using DCFDA, a fluorogenic dye that assesses ROS within the cell. As shown in Figure 3A, carvacrol exposure significantly upregulated the levels of ROS in HOS and U-2 OS cells. Furthermore, coadministration of N-acetyl-L-cysteine (NAC), a ROS scavenger, resulted in attenuation of the increased cleaved caspase 3 and PARP in these cells ( Figure 3B). The results suggest that ROS may be involved in carvacrol-induced apoptosis in osteosarcoma HOS and U-2 OS cells. The cells were exposed to carvacrol in the presence or absence of N-acetyl-L-cysteine (NAC) for 24 h, and ROS levels were then measured by flow cytometry using 2′,7′-dichlorofluorescin diacetate (DCFDA) staining. Data are presented as mean ± S.E.M. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared between groups. (B) Attenuation of ROS levels by NAC suppresses the levels of cleaved caspase 3 and cleaved PARP in carvacrol-exposed osteosarcoma cell lines. The cells were exposed by flow cytometry using 2 ,7 -dichlorofluorescin diacetate (DCFDA) staining. Data are presented as mean ± S.E.M. of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 when compared between groups. (B) Attenuation of ROS levels by NAC suppresses the levels of cleaved caspase 3 and cleaved PARP in carvacrol-exposed osteosarcoma cell lines. The cells were exposed to carvacrol in the presence or absence of NAC for 24 or 48 h. After that, the cells were analyzed by Western blotting using indicated antibodies by independent duplicate experiments.

Attenuation of ER Stress Enhances the Antitumor Activity of Carvacrol in Osteosarcoma Cell Lines
In this report, we also observed that carvacrol exposure resulted in increased expression of ER stress markers, including GRP78, IRE1a, PERK, and CHOP, in osteosarcoma HOS and U-2 OS cells (Figure 4). This indicates carvacrol could have the ability to trigger ER stress in osteosarcoma cell lines. However, ER stress has been found to both protect cells from apoptosis and to activate apoptosis in these cells [35]. To determine the roles of ER stress in carvacrol-induced apoptosis, 4-PBA, an ER stress inhibitor, was coadministrated in these cells. As shown in Figure 5A, coadministration of 4-PBA at a higher dose (3 mM) resulted in attenuation of ER stress markers and simultaneously increased the levels of cleaved caspase-3 and cleaved PARP. In addition, coadministration of 4-PBA further suppressed clonogenic activity in carvacrol-exposed osteosarcoma cell lines ( Figure 5B). The results indicate that ER stress triggered by carvacrol exposure protect cells from apoptosis and its attenuation enhances the antitumor activity of carvacrol in osteosarcoma cell lines. to carvacrol in the presence or absence of NAC for 24 or 48 h. After that, the cells were analyzed by Western blotting using indicated antibodies by independent duplicate experiments.

Attenuation of ER Stress Enhances the Antitumor Activity of Carvacrol in Osteosarcoma Cell Lines
In this report, we also observed that carvacrol exposure resulted in increased expression of ER stress markers, including GRP78, IRE1a, PERK, and CHOP, in osteosarcoma HOS and U-2 OS cells (Figure 4). This indicates carvacrol could have the ability to trigger ER stress in osteosarcoma cell lines. However, ER stress has been found to both protect cells from apoptosis and to activate apoptosis in these cells [35]. To determine the roles of ER stress in carvacrol-induced apoptosis, 4-PBA, an ER stress inhibitor, was coadministrated in these cells. As shown in Figure 5A, coadministration of 4-PBA at a higher dose (3 mM) resulted in attenuation of ER stress markers and simultaneously increased the levels of cleaved caspase-3 and cleaved PARP. In addition, coadministration of 4-PBA further suppressed clonogenic activity in carvacrol-exposed osteosarcoma cell lines ( Figure  5B). The results indicate that ER stress triggered by carvacrol exposure protect cells from apoptosis and its attenuation enhances the antitumor activity of carvacrol in osteosarcoma cell lines.  HOS or U-OS cells were exposed to carvacrol for 24 h in the presence or absence of 4-PBA. After that, the exposed cells were analyzed by Western blotting using indicated antibodies by independent duplicate experiments. (B) Attenuation of ER stress suppresses the colony formation in carvacrol-exposed cells. The cells were exposed to carvacrol for 16 h in the presence or absence of 4-PBA. After that, the exposed cells were plated in colony formation assay, and the number of colonies was counted. Results are presented as mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 compared to the 0 mM group.

Discussion
Various anticancer compounds show the ability to trigger endoplasmic reticulum (ER) stress. The stimulated ER stress either enhances cancer cell death or protects cells from apoptosis, which acts as a mechanism of resistance to chemotherapy. For instance, α-mangostin, a natural compound isolated from mangosteen, induces apoptosis and ER stress in osteosarcoma cell lines. Cotreatment with 4-PBA (an ER stress inhibitor) HOS or U-OS cells were exposed to carvacrol for 24 h in the presence or absence of 4-PBA. After that, the exposed cells were analyzed by Western blotting using indicated antibodies by independent duplicate experiments. (B) Attenuation of ER stress suppresses the colony formation in carvacrolexposed cells. The cells were exposed to carvacrol for 16 h in the presence or absence of 4-PBA. After that, the exposed cells were plated in colony formation assay, and the number of colonies was counted. Results are presented as mean ± S.E.M. (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 compared to the 0 mM group.

Discussion
Various anticancer compounds show the ability to trigger endoplasmic reticulum (ER) stress. The stimulated ER stress either enhances cancer cell death or protects cells from apoptosis, which acts as a mechanism of resistance to chemotherapy. For instance, αmangostin, a natural compound isolated from mangosteen, induces apoptosis and ER stress in osteosarcoma cell lines. Cotreatment with 4-PBA (an ER stress inhibitor) suppresses αmangostin-induced apoptosis in these cells, suggesting that α-mangostin-induced ER stress positively contributes to apoptosis in these cells [36]. On the contrary, coadministration of 4-PBA reduces the expression of GRP78 and CHOP and enhances apoptosis in cisplatintreated osteosarcoma cell lines, indicating that cisplatin-induced ER stress protects cell from apoptosis in these cell lines [37]. Khan et al. showed that carvacrol nanoemulsion could induce apoptosis and ER stress in lung adenocarcinoma A549 cells [29]. However, the link between apoptosis and ER stress in carvacrol-treated lung cancer cells is unclear. In this report, our data demonstrated that carvacrol could have the potential for triggering ER stress in osteosarcoma cell lines and that coadministration of the ER stress inhibitor 4-PBA enhanced carvacrol-induced apoptosis in osteosarcoma cell lines, indicating that upregulated ER stress protects osteosarcoma cells from carvacrol cytotoxicity.
Reactive oxygen species (ROS) generation has been linked to ER stress. ROS can be produced in ER as byproducts of protein folding, and ER stress in some conditions upregulates the levels of ROS in the ER or mitochondria. In addition, overwhelming ROS levels have been shown to influence ER protein folding and induce ER stress [38]. Therefore, it is suggested that ROS generation could be downstream or upstream of ER stress. In this report, we demonstrated that carvacrol exposure induced ER stress and increased ROS levels in osteosarcoma cell lines, but the interaction between ER stress and ROS needs to be further investigated in these cells.
The literature shows that various concentrations of carvacrol could induce cytotoxic effect in different cancer cells. It was found that 0.5 to 1 mM of carvacrol significantly suppressed cell growth in the nonsmall lung cancer cell line A549 [39], while 0.3 to 0.7 mM of carvacrol induced cell cycle arrest and apoptosis in the colon cancer cell lines HCT116 and LoVo [22]. In addition, higher than 0.3 mM carvacrol significantly induced apoptosis in several breast cancer cell lines, including BT-474, MDA-MB-231, and MCF-7 cells [40]. In this report, the data showed that 0.3 to 1 mM of carvacrol was effective concentration to inhibit proliferation of osteosarcoma HOS and U-2 OS cell lines. Recently, some potential ways to enhance antitumor activity of carvacrol have been revealed, such as nanoemulsions for drug delivery [29] and coexposure to 5-FU [41] or X-radiation [42]. Based on our results, ER stress may be a target to enhance antitumor activity of carvacrol in osteosarcoma cell lines.
Several studies have successfully demonstrated the antitumor activities of carvacrol in in vivo experiments. For example, Sivaranjani et al. showed that administration of carvacrol significantly suppressed the tumor incidence and the number of colonic aberrant crypt foci in DMH (1,2-dimethylhydrazine)-induced colon carcinogenesis in male rats [43]. Subramaniyan et al. further demonstrated that carvacrol exposure suppressed the elevation of tumor marker levels and reduced mast cell density and cell proliferation in a DEN (diethylnitrosamine)-induced hepatocarcinogenesis rat model [44]. In addition, a carvacrol nanoemulsion was found to demonstrate anticancer activity against human lung adenocarcinoma A549 cell line in an in vivo xenograf mice model [29]. In this report, our data revealed that carvacrol significantly suppressed cell viabilities of osteosarcoma HOS and U-2 OS cell lines, and the inhibition of ER stress by 4-PBA enhanced its anticancer activity in in vitro studies. Whether these effects could be verified in in vivo assays should be further addressed in the future. Furthermore, carvacrol should be compared to other standard drugs, such as doxorubicin or cisplatin, at the same time to understand the efficacy of drug treatment.
Several studies have demonstrated that poor chemotherapy response is correlated with poor prognosis in osteosarcoma patients [4,45]. In addition, tumor relapse is a difficult issue for treating osteosarcoma. Once relapse occurs, the five-year post-relapse overall survival rate is very low at approximately 20 to 30% [7,8]. Moreover, in several reported studies, chemotherapy used for treating the first relapse was not associated with survival after recurrence [7,9,10]. Therefore, it is urgently necessary to develop effective drugs for patients suffering from osteosarcoma. Some anticancer drugs have been evaluated for treating osteosarcoma in clinical trials, such as pemetrexed and carboplatin [46]. In this report, we demonstrated the antitumor activity of carvacrol against osteosarcoma and revealed that the inhibition of carvacrol-induced ER stress by 4-PBA enhanced the antitumor activity of carvacrol. We hope our findings can contribute to the further development of carvacrol as anticancer drugs for treating osteosarcoma.
In conclusion, this study showed that carvacrol exposure significantly inhibited cell viabilities, increased ROS generation, and resulted in apoptosis in osteosarcoma cell lines. In addition, we found that carvacrol-induced ER stress protected cells from apoptosis and that its attenuation enhanced the antitumor activity of carvacrol in osteosarcoma cell lines. These results provide an insight into the mechanism of carvacrol action and suggest potential therapeutic strategies for osteosarcoma.